Scrub typhus is a neglected rickettsial disease that threatens one billion inhabitants of the Asia-Pacific region and causes one million new cases annually. The infection can rapidly progress to severe morbidity and has a mortality rate of up to 50%. The etiologic agent of scrub typhus is Orientia tsutsugamushi (Ot), a mite- transmitted obligate intracellular bacterium that invades leukocytes and endothelial cells. Despite its global health impact, the bacterial factors that enable Ot to replicate in the cytosol without igniting th immune response and modulate host cell functions so that it can survive and cause disease are undefined. Ankyrin repeat-containing proteins (Anks) are microbial virulence factors that bind specific host proteins to co-opt or subvert eukaryotic functions to the pathogens' benefit. Ot encodes one of the largest Ank arsenals of any bacterium. Maintenance of this large number of over Anks the course of its reductive evolution implies their importance to Ot virulence. Elucidating Ank functions is therefore central to understanding Ot pathobiology. Ot Anks are Type 1 secretion system effectors. We identified four (Ank1, Ank4, Ank6, Ank9) that Ot expresses as it establishes infection, determined the host cell processes that they modulate, and identified their host cell interacting partners. NF-?B is essential for defense against pathogens. Translocation of the NF-?B dimer, p50/p65 into the nucleus activates transcription of hundreds of antimicrobial genes. We made the exciting discoveries that (i) Ot thwarts the NF-?B response by blocking p65 accumulation in the nucleus and by inhibiting NF-?B dependent transcription, (ii) p65 accumulation in the nucleus is blocked in cells ectopically expressing Ank1 or Ank6, and (iii) Ank1 and Ank6 each interact with p65. In Aim 1, we will define how Ank1 and Ank6 retard p65 accumulation in the nucleus, confirm if each inhibits p50/p65-driven transcription, and determine how each binds p65. We also discovered that Ot blocks host cell protein secretion and induces endoplasmic reticulum (ER) stress, a cellular response that can lead to apoptosis. Ank4 and Ank9 contribute to these phenomena, as ectopically expressed Ank4 and Ank9 inhibit protein secretion and evoke ER stress. GFP-Ank4 traffics to and disrupts the morphology of the ER. GFP-Ank9 traffics to the Golgi, retrograde traffics to the ER, and destabilizes the Golgi and ER. We identified BAT3 and COPB2 as Ank4 and Ank9 interacting partners, respectively. BAT3 is a chaperone that mediates ER stress-induced apoptosis. COPB2 directs Golgi- to-ER retrograde trafficking and intracellular transport. In Aim 2, we will define how Ank4 and Ank9 modulate the secretory pathway, benefit Ot fitness, inhibit ER stress-induced apoptosis, and dissect how their interactions with BAT3 and COPB2 mediate these phenomena. Completing these Aims will greatly advance understanding of how Ot modulates cellular processes to establish a permissive niche and will add to the knowledge of the diverse ways by which intracellular bacteria subvert NF-?B and manipulate the secretory pathway.